Continuous culture apparatus with mobile vessel, allowing selection of fitter cell variants and producing a culture in a continuous manner

ABSTRACT

A method and device for growing plant, animal or stem cells in a continuous manner.

FIELD OF THE INVENTION

The described invention provides a method and a device that allowselection of living cells, with increased rates of reproduction andspecific metabolic properties, in a liquid or semi-solid medium. For theprocess of selection (adaptive evolution), genetically variant organisms(mutants) arise in a population and compete with other variants of thesame origin. Those with the fastest rate of reproduction increase inrelative proportion over time, leading to a population (and individualorganisms) with increased reproductive rate. This process can improvethe performance of organisms used in industrial processes or academicpurpose. The present invention utilizes a continuous culture apparatusto achieve the viable production of living cells, for example, yeast,plant cells, animal cells or stem cells. The present invention may beused to produce an active ingredient or biologic that is produced by theliving cells. The active ingredient or biologic may in turn be used as adiagnostic, preventive, or therapeutic agent.

BACKGROUND OF THE INVENTION

Selection for increased reproductive rate (fitness) requires sustainedgrowth, which is achieved through regular dilution of a growing culture.In the prior art this has been accomplished two ways: serial dilutionand continuous culture, which differ primarily in the degree ofdilution.

Serial culture involves repetitive transfer of a small volume of grownculture to a much larger vessel containing fresh growth medium. When thecultured cells have grown to saturation in the new vessel, the processis repeated. This method has been used to achieve the longestdemonstrations of sustained culture in the literature (Lenski &Travisano: Dynamics of adaptation and diversification: a10,000-generation experiment with bacterial populations. 1994. Proc NatlAcad Sci USA. 15:6808-14), in experiments which clearly demonstratedconsistent improvement in reproductive rate over period of years. Thisprocess is usually done manually, with considerable labor investment,and is subject to contamination through exposure to the outsideenvironment. Serial culture is also inefficient, as described in thefollowing paragraph.

The rate of selection, or the rate of improvement in reproductive rate,is dependant on population size (Fisher: The Genetical Theory of NaturalSelection.1930. Oxford University Press, London, UK). Furthermore, in asituation like serial transfer where population size fluctuates rapidly,selection is proportional to the harmonic mean (N) of the population(Wright: Size of population and breeding structure in relation toevolution. 1938. Science 87: 430-431), and hence can be approximated bythe lowest population during the cycle.

Population size can be sustained, and selection therefore made moreefficient, through continuous culture. Continuous culture, asdistinguished from serial dilution, involves smaller relative volumesuch that a small portion of a growing culture is regularly replaced byan equal volume of fresh growth medium. This process maximizes theeffective population size by increasing its minimum size during cyclicaldilution. Devices allowing continuous culture are termed “chemostats” ifdilutions occur at specified time intervals, and “turbidostats” ifdilution occur automatically when the culture grows to a specificdensity.

For the sake of simplicity, both types of devices will hereafter begrouped under the term “chemostat”. Chemostats were inventedsimultaneously by two groups in the 1950's (Novick & Szilard:Description of the chemostat. 1950. Science 112: 715-716) and (Monod: Latechnique de la culture continue—Théorie et applications.1950. Ann.Inst. Pasteur 79:390-410). Chemostats have been used to demonstrateshort periods of rapid improvement in reproductive rate (Dykhuizen DE.Chemostats used for studying natural selection and adaptiveevolution.1993. Methods Enzymol. 224:613-31).

Traditional chemostats are unable to sustain long periods of selectionfor increased reproduction rate, due to the unintended selection ofdilution-resistant (static) variants. These variants are able to resistdilution by adhering to the surface of the chemostat, and by doing so,outcompete less sticky individuals including those that have higherreproductive rates, thus obviating the intended purpose of the device(Chao & Ramsdell: The effects of wall populations on coexistence ofbacteria in the liquid phase of chemostat cultures,. 1985. J. Gen.Microbiol. 131: 1229-36).

One method and chemostatic device (the Genetic Engine) has been inventedto avoid dilution resistance in continuous culture (patent U.S. Pat. No.6,686,194-B1 filed by PASTEUR INSTITUT [FR] & MUTZEL RUPERT [DE]) . Thismethod uses valve controlled fluid transfer to periodically move thegrowing culture between two chemostats, allowing each to be sterilizedand rinsed between periods of active culture growth. The regularsterilization cycles prevent selection of dilution-resistant variants bydestroying them. This method and device achieves the goal, but requiresindependent complex manipulations of several fluids within a sterile(sealed) environment, including one (NaOH) which is both very causticand potentially very reactive, quickly damaging valves, and posingcontainment and waste-disposal problems. The chemostatic device is alsolimited in that no provisions are made to provide a support for cells togrow on

There are some types of cells that are difficult to culture in largeamounts due to the conditions the cells require to survive and grow. Itis believed that these cells could grow in conditions provided by acontinuous culture approach. This is particularly the case for stemcells.

For example, human embryonic stem cells are typically grown by isolatingand transferring a stem cell mass into a plastic laboratory culture dishthat contains a nutrient broth known as culture medium. The cells divideand spread over the surface of the dish. The inner surface of theculture dish is typically coated with mouse embryonic skin cells thathave been treated so they will not divide. This coating layer of cellsis called a feeder layer. The reason for having the feeder layer in thebottom of the culture dish is to give the human embryonic stem cells asticky surface to which they can attach. Also, the feeder cells releasenutrients into the culture medium. Recently, scientists have begun todevise ways of growing embryonic stem cells without the mouse feedercells. This is a significant scientific advancement because it avoidsthe risk that viruses or other macromolecules in the mouse cells may betransmitted to the human cells.

Over the course of several days, the cells of the inner cell massproliferate and begin to crowd the culture dish. When this occurs, theyare removed gently and plated into several fresh culture dishes. Theprocess of replating the cells is repeated many times and for manymonths, and is called subculturing. Each cycle of subculturing the cellsis referred to as a passage. After six months or more, the originalcells of the cell mass yield millions of embryonic stem cells. Embryonicstem cells that have proliferated in cell culture for six or more monthswithout differentiating, are pluripotent, and appear genetically normalare referred to as an embryonic stem cell line.

Once cell lines are established, or even before that stage, batches ofthem can be frozen and shipped to other laboratories for further cultureand experimentation. However, continuous culture grants the advantage ofsuppressing a maximum of manipulations that stress the living cells andcreate a potential source of contamination. When a culture is started,continuous culture conditions allow the skilled artisan to takeadvantage of a continuous production of cells. Once stem cells are beingproduced, the production of stems cells could continue withoutinterruption to produce substantially more stem cells than methods thatare typically used today.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide animproved (and completely independent) method and device for continuousculture of cells (including bacteria, archaea, eukaryotes and viruses)without interference from dilution-resistant variants. Like otherchemostats, the device provides a means for regular dilution of a grownculture with fresh growth medium, a means for gas exchange between theculture and the outside environment, sterility, and automatic operationas either a chemostat or a turbidostat.

Additionally, it is an object of the present invention to provide animproved and distinct method and device for continuous culture of cellssuch as plant cells, animal cells or stem cells. Stem cells that may becultured with the present invention include but are not limited toembryonic stem cells, fetal stem cells, umbilical cord stem cells,placenta derived stem cells, and adult stem cells. The adult stem cellsthat may be cultured with the present invention include but are notlimited to hematopoietic stem cells, bone marrow stem cells, stromalcells, astrocytes and oligidendrrocytes (e.g, Hematopoietic Stem CellProtocols by C. Klug and C. Jordan, Humana Press, Totowa, N.J., 2002,incorporated by reference herein).

The present invention is designed to achieve these goals without anyfluid transfer, including sterilization or rinsing functions. Thisrepresents a specific advantage of the present invention with respect toprior art in so far as it avoids the hazards and difficulties associatedwith sterilization and rinsing, including containment and complex fluidtransfers involving caustic solvents.

Continuous culture is achieved inside a flexible sterile tube filledwith growth medium. The medium and the chamber surface are static withrespect to each other, and both are regularly and simultaneouslyreplaced by peristaltic movement of the tubing through “gates”, orpoints at which the tube is sterilely subdivided by clamps that preventthe cultured cells from moving between regions of the tube. UV gates canalso (optionally) be added upstream and downstream of the culture vesselfor additional security.

The present method and device are also an improvement over prior artinsofar as they continually, rather than periodically, select againstadherence of dilution-resistant variants to the chemostat surfaces, asreplacement of the affected surfaces occurs in tandem with the processof dilution.

The tube is subdivided in a transient way such that there are regionsthat contain saturated (fully grown) culture, regions that contain freshmedium, and a region between these two, wherein one or more chambersreferred to as growth or culture chambers are present to form a growthchamber region in which grown culture is mixed with fresh medium toachieve dilution. The gates are periodically released from one point onthe tube and replaced at another point, such that grown culture alongwith its associated growth chamber surface and attached static cells, isremoved by isolation from the growth chamber and replaced by both freshmedium and fresh chamber surface.

BRIEF DESCRIPTION OF THE DRAWINGS

Without being exhaustive and limiting, one possible generalconfiguration will include several components as described hereafter. Inthe following the present invention is exemplarily explained on thebasis of a preferred embodiment, thereby referring to the drawings inwhich:

FIG. 1 displays an overall view of a possible configuration of thedevice in which:

(1) represents the flexible tubing containing the different regions ofthe device which are: upstream fresh medium (7), growth chamber (10),sampling chamber (11) and disposed grown culture region (15)

(2) represents the thermostatically controlled box allowing regulationof temperature according to conditions determined by user, and in whichmay be located:

-   -   a. said growth chamber (10),    -   b. said sampling chamber (11),    -   c. upstream gate (3) defining the beginning of said growth        chamber (10),    -   d. downstream gate (4) defining the end of said growth chamber        (10) and the beginning of said sampling chamber (11)    -   e. second downstream gate (5) defining the end of said sampling        chamber (11),    -   f. turbidimeter (6) allowing the user or automated control        system to monitor optical density of growing culture and to        operate a feedback control system (13), allowing controlled        movement of the tubing (1) on the basis of culture density        (turbidostat function),    -   g. one or several agitators (9).

It should be noted that the device elements listed in a-g may also belocated outside

of, or in the absence of, a thermostatically controlled box.

(7) represents the fresh medium in unused flexible tubing,

(8) represents a barrel loaded with fresh medium filled tubing, in orderto dispense said fresh medium and tubing during operations.

(12) represents optional ultra-violet radiation gates,

(13) represents the control system that can consist of a computerconnected with means of communication to different monitoring oroperating interfaces, like optical density turbidimeters, temperaturemeasurement and regulation devices, agitators and tilting motors, etc,that allow automation and control of operations,

(14) represents the optional disposal barrel on which to wind up tubingcontaining disposed grown culture filled tubing,

(15) represents disposed grown culture located downstream of saidsampling chamber.

FIG. 2 displays two possible positions of the device, exemplifying thefact that said thermostatically controlled box (2) and other pieces ofsaid device associated with said culture chamber can be tilted tovarious degrees for agitation purposes, gas circulation and removalpurposes, and purposes of guaranteeing the removal of granulated(aggregated) cells that might escape dilution by settling to the bottom.

FIGS. 3 to 9 represents said flexible tubing (1) in place in saidthermostatically controlled box (2) and introduced through gates (3),(4) and (5) through which said tubing will stay during all steps ofprocess and through which said tubing will move according to itsperistaltic movement.

FIG. 3 symbolizes status T0 of the device in which all regions of saidflexible tubing are filled with fresh medium before injection of thecell intended for continuous culture.

FIG. 4 symbolizes status T1 of said flexible tubing just after injectionof cell strain.

FIG. 5 symbolizes status T2 of the device which is a growing periodduring which the culture grows in the region defined as the growthchamber (10) limited by said gates (3) and (4).

FIG. 6 symbolizes status T3 of device, just after the first peristalticmovement of tubing and associated medium, which determines the beginningof the second growing cycle, introducing fresh tubing and medium throughmovement of gate 3 simultaneous with a transfer of equivalent volume oftubing, medium, and grown culture out of the growth chamber region (10)and into the sampling chamber region (11) by movement of gate 4. It iscritical to recognize that the tubing, the medium that is within thetubing, and any culture that has grown in that medium, all movetogether. Fluid transfer only occurs insofar as fresh medium and grownculture mix together through agitation within the growth chamber region.

FIG. 7 symbolizes status T4 of the device which is the second growingcycle; during this cycle cells that remain in the growth chamber afterperistaltic movement of the tubing can now grow using nutrients providedin the fresh medium that is mixed with the remaining culture during thisstep.

FIG. 8 symbolizes status T5 of device, just after the second peristalticmovement of the tubing and the contained medium, which determines thebeginning of the third growing cycle, introducing fresh tubing andmedium through movement of gate 3 simultaneous with a transfer ofequivalent volume of tubing, medium, and grown culture out of the growthchamber region (10) and into the sampling chamber region (11) bymovement of gate 4.

FIG. 9 symbolizes status T6 of device which is the third growing cycle;this step is equivalent to status T4 and indicates the repetitive natureof further operations. Samples of selected cells may be removed at anytime from the sampling chamber region (11) using a syringe or otherretrieval device.

FIG. 10 displays a possible profile of teeth determining a gate in theconfiguration which consists of two stacking teeth pinching flexibletubing. Gates could also be determined by single teeth pressing againsta moveable belt, removable clamps, or other mechanisms that preventmovement of cells through the gate and which can be alternately placedand removed in variable positions along the tubing.

DETAILED DESCRIPTION OF INVENTION

The basic operation of the device is depicted in FIGS. 3 through 9.

One potential configuration for the present device is shown in FIG. 1,as it appears after having been loaded with a fresh tube of sterilemedium (shown divided into regions A-H by said gates (3), (4) and (5)).

Inoculation of the device with the chosen cell could be achieved byintroduction of the cell into the growth chamber (FIG. 3), throughinjection (FIG. 4, region B). The culture would then be allowed to growto the desired density and continuous culture would begin (FIG. 5).

Continuous culture would proceed by repetitive movements of the gatedregions of tubing. This involves simultaneous movements of the gates,the tubing, the medium, and any culture within the tubing. The tubingwill always move in the same direction; unused tubing containing freshmedium (and hereafter said to be ‘upstream’ of the growth chamber (7))will move into the growth chamber and mix with the culture remainingthere, providing the substrate for further growth of the cells containedtherein. Before introduction into the growth chamber region, this mediumand its associated tubing will be maintained in a sterile condition byseparation from the growth chamber by the upstream gates (3). Usedtubing containing grown culture will simultaneously be moved‘downstream’ and separated from the growth chamber by the downstreamgates (4).

When one or more growth chambers are present, the growth chambers may beused for the same or different purpose. For example, living cells couldbe grown in a first growth chamber and a second growth chamber with thesame or different conditions. In one embodiment, a first growth chambercan be used to grow cells and a second growth chamber may then be usedto treat the living cells under different conditions. For example, thecells may be treated to induce the expression of a desired product.Components or additives of the culture medium itself may be added priorto or after the culture begins. For example, all components or additivescould be included in the media before beginning the culture, orcomponents can be injected into one or more of the growth chambers afterthe culture has been initiated.

Gate configuration is not a specific point of the present patentapplication. For example, in a given configuration, gates can bedesigned through one chain of multiple teeth simultaneously moved or inanother configuration separated in distinct synchronized chains asdepicted in FIG. 1. Gates can consist of a system made of two teethpinching the tubing in a stacking manner as described in FIG. 10,avoiding contamination between regions G and H of the tubing through theprecision of the interface between the teeth. In another configuration,sterile gates can be obtained by pressing one tooth against one side ofthe tubing and thereby pressing the tubing tightly against a fixedchassis along which tubing is slid during its peristaltic movement, assketched in FIG. 3 to 9, marks 3, 4 and 5.

Said thermostatically controlled box (2) is obtained by already knownmeans such as a thermometer coupled with a heating and cooling device.

Aeration (gas exchange), when required for growth of the cultured cellor by the design of the experiment, is achieved directly and withoutmechanical assistance by the use of gas permeable tubing. For exampleand without being limiting, flexible gas permeable tubing can be made ofsilicone. Aeration could be achieved through exchange with the ambientatmosphere or through exchange with an artificially defined atmosphere(liquid or gas) that contacts the growth chamber or the entirechemostat. When an experiment demands anaerobiosis the flexible tubingcan be gas impermeable. For example and without being limiting, flexiblegas impermeable tubing can be made of coated or treated silicone.

For anaerobic evolution conditions, regions of the tubing can also beconfined in a specific and controlled atmospheric area to control gasexchange dynamics. This can be achieved either by making saidthermostatically controlled box gastight and then injecting neutral gasinto it or by placing the complete device in an atmosphere controlledroom.

Counter-selection of static variants is achieved by replacement of thegrowth chamber surface along with growth medium.

The device is further designed to be operable in a variety oforientations with respect to gravity, that is, to be tilted as shown byFIG. 2, along a range of up to 360°.

Dilution-resistant variants may avoid dilution by sticking to oneanother, rather than to the chamber wall if aggregated cells can fallupstream and thereby avoid removal from the chamber. Hence it isdesirable that the tubing generally be tilted downward, such thataggregated cells will fall toward the region that will be removed fromthe growth chamber during a cycle of tube movement. This configurationinvolves tilting the device so that the downstream gates are below theupstream gates with respect to gravity.

The growing chamber can be depressurized or over pressurized accordingto conditions chosen by the experimenter. Different ways of adjustingpressure can be used, for instance applying vacuum or pressurized air tothe fresh medium and tubing through its upstream extremity and acrossthe growth chamber; another way of depressurizing or overpressurizingtubing can be done by alternate pinching and locking tubing upstream ofor inside the growth chamber.

When the medium is contained in gas permeable tubing, air bubbles mayform within the medium. These will rise to the top of a sealed region oftubing and become trapped there until the movement of the region (andthe gates defining it) releases the region into either the growthchamber, the sampling chamber or the endpoint of the chemostat (FIGS. 6,regions D-C, B or A, respectively). If the device is tilted downwardsuch bubbles will accumulate in the growth chamber or sampling chamberand displace the culture. The device is designed to periodically tiltupward for a cycle of the tube movement, allowing for the removal ofaccumulated gas from said chambers.

Tilting movements of the device, and/or shaking of the growth chamber byan external device (9) can be used to decrease aggregation of cellswithin the growth chamber. Alternatively, one or several stirring barscan be included in the tubing filled with fresh medium beforesterilization and magnetically agitated during culture operations.

The proportional length of the regions of fresh medium defined by theupstream gates as compared to the length of the culture chamber willdefine the degree of dilution achieved during a cycle.

The frequency of dilution can be determined either by timing (chemostatfunction) or by feedback regulation whereby the density of the culturein the growth chamber is measured by a turbidimeter (FIG. 1—mark 6) andthe dilution cycle occurs when the turbidity reaches a threshold value(turbidostat function).

The sampling chamber allows withdrawing grown culture in order toanalyze the outcome of an experiment, collect cells with improved growthrate for further culture, storage, or functional implementation, orother purposes such as counting the population, checking the chemicalcomposition of the medium, or testing the pH of grown culture. In orderto achieve permanent monitoring of pH inside growth chamber, tubing caninclude by construction a pH indicator line embedded/encrusted in thewall of the tubing.

Any form of liquid or semi-solid material can be used as a growth mediumin the present device. The ability to utilize semi-solid growthsubstrates is a notable advancement over prior art. The growth medium,which will define the metabolic processes improved by the selectionprocess, can be chosen and defined by the user.

If needed, this device can contain multiple growth chambers, such thatthe downstream gates of one growth chamber become the upstream gates ofanother. This could, for example, allow one cell to grow alone in thefirst chamber, and then act as the source of nutrition for a second cell(or virus) in the second chamber.

The invention may be used to produce a preparation, such as a biologicfor a drug, a vaccine, or an antitoxin, that is synthesized from cellsgrown by the invention or their products. The biologic may be used as adiagnostic, preventive, or therapeutic agent. For example, the presentinvention may be used to produce therapeutic proteins such as insulin.

In a preferred embodiment, the device and/or method may be cycled in amanner to continually collect stem cells in their undifferentiatedstate. Furthermore, the culture conditions may be modified to inhibitthe differentiation of the stem cells. For example, stem celldifferentiation inhibitors (e.g., inhibitors of aldehyde dehydrogenase,inhibitors of phosphoinositide 3-kinase, TGF Receptor Kinase inhibitors,TGF-B Receptor Kinase Inhibitor etc. . . . ) may be added to the culturemedium. Alternatively, process conditions such as the amount of oxygendelivered to the culture medium may be increased or decreased to improvethe growth of certain stem cells and/or slow down or improvedifferentiation of the stem cells.

As some cells require a substrate to grow, a physical support orstructure can be added to the vessel culture chamber. In a preferredembodiment, a continuous support could be added inside the tubing like acontinuous fiber bed, constituted by a thin continuous fiber likesupport structure, can be added to the vessel culture chamber whichcould let cells grow in 3 dimensions. For example, the support could bea fiber bed, which provides support for the growth of cells such as stemcells, plants cells and other types of cells that prefer such a supportstructure, and in some specific conditions or change of conditions, toprocess the natural selection for targeted mutations.

In a preferred embodiment, a fibrous material as described in Huang etal., Continuous Production of butanol by Clostridium acetobutylicumimmobilized in a fibrous bed reactor, Appl Biochem Biotechnol. 2004Spring; 113-116:887-98, incorporated by reference herein. The structureand size of the tubing may also be varied to avoid the need forincorporating a support structure into the mobile vessel culturechamber. In a preferred embodiment, tubing with a smaller diameter isused so the cells may adhere in a more natural manner.

This device and method allows researchers and product developers toevolve any strain of culturable living cells in suspension or any strainof culturable living cells which are not in suspension which grow on thewall of the tubing or on a support which could be a fiber bed in thetubing through sustained growth (continuous culture); the resultingimproved cell can constitute a new strain or species. These new cellscan be identified by mutations acquired during the course of culture,and these mutations may allow the new cells to be distinguished fromtheir ancestors' genotype characteristics. This device and method allowthe researcher to select new strains of any living cell by segregatingindividuals with improved rates of reproduction through the process ofnatural selection. The invention also provides an improved andcompletely distinct method and device for continuous culture of cellssuch as yeast, plant cells, animal cells or stem cells.

In a further embodiment, an emitter can be used to subject the cells,permanently or temporarily, to at least one of radio waves, light waves,x-rays, sound waves, an electro magnetic field, a radioactive field,radioactive media, or combinations thereof. The following publicationsare incorporated by reference: Biofizika. 2005 July-August,50(4):689-92; Bioelectromagnetics. 2005 September, 26(6):431-9; ChemCommun (Camb). Jan. 14, 2005, (2):174-6; Biophys J. 2005 February,88(2):1496-9; Bioelectromagnetics. 1981, 2(3):285-9; Sb Lek. 1998,99(4):455-64; Antimicrob Agents Chemother. 2004 December, 48(12):4662-4;J Food Prot. 2003 September, 66(9):1712-5; Astrobiology. 2006 April,6(2):332-47; Life Sci Space Res. 1970, 8:33-8; Adv Space Res. 1995March, 15(3):211-4; Radiat Res. 2006 May, 165(5):532-7; Mutagenesis.2004 September, 19(5):349-54; Cancer Sci. 2006 June, 97(6):535-9; ApplEnviron Microbiol. 2006 May, 72(5):3608-14; and Pol J Microbiol. 2005,54 Suppl:7-11.

In another embodiment, the growth chamber region of the device could besubjected to, permanently or temporarily, subjecting the cells to adifferent gravitational force. For example, the cells can be grown in amicrogravity environment. The following publications are incorporated byreference: J Gravit Physiol. 2004 March;11(1):75-80; Immunol Rev. 2005Dec;208:267-80; and J Gravit Physiol. 2004 July;11(2):P181-3.

Modifications and variations of the present invention relating to theapparatus and method will be obvious to those skilled in the art fromthe foregoing detailed description of the invention. Such modificationsand variations are intended to come within the scope of the appendedclaims.

1. A device for growing living cells in a continuous manner, comprising:a flexible tubing containing culture medium and a surface in which theliving cells can grow on; and a system of clamps, each capable of openand closed positions, the clamps being positioned so as to be able todivide the tubing into: i) an upstream region containing unused culturemedium; ii) a downstream region containing spent culture medium; andiii) a growth chamber region for growing said cells disposed between theupstream and downstream regions; wherein the system of clamps isconstructed and arranged to open and close so as to clamp off and definethe growth chamber region of the tubing between the upstream anddownstream regions of the tubing, and to cyclically redefine the growthchamber region of the tubing so that a first portion of the previouslydefined growth chamber region becomes a portion of the downstream regionof the tubing, and a portion of the previously defined upstream regionof the tubing becomes a portion of the growth chamber region of thetubing.
 2. The device according to claim 1, wherein the system of clampsis structured and arranged so that each of the clamps does not move withrespect to the tubing when said clamp is in the closed position.
 3. Thedevice according to claim 1, wherein the surface is the interior surfaceof the tubing.
 4. The device according to claim 3, wherein the surfaceis a continuous support inserted inside the tubing.
 5. The deviceaccording to claim 4, wherein the means for providing a surface is acontinuous fiber.
 6. The device according to claim 1, wherein the tubingis gas permeable
 7. The device according to claim 1, wherein the tubingis gas impermeable.
 8. The device according to claim 1, wherein thetubing is one of transparent and translucent to permit a turbidimeter todetermine the density of the culture.
 9. The device according to claim1, wherein the device further comprises a pressure regulator constructedto change a pressure of the growth chamber portion of the tubingrelative to ambient pressure.
 10. The device according to claim 1,wherein the tubing comprises a pH indicator.
 11. The device according toclaim 1, further comprising a temperature regulator constructed to allowcontrol of a temperature of the growth chamber region of the tubing. 12.The device according to claim 1, wherein the device further comprises anagitator constructed to allow agitation of the growth chamber portion ofthe tubing.
 13. The device according to claim 12, wherein the agitatorcomprises at least one stirring bar.
 14. The device according to claim1, further comprising an emitter constructed to subject the growthculture chamber region to at least one of radio waves, light waves,x-rays, sound waves, an electro magnetic field, and a radioactive field.15. The device according to claim 1, further comprising a means forsubjecting the growth chamber region to a different gravitational force.16. The device according to claim 1, wherein said growth chamber regioncomprises one or more growth chambers containing culture medium.
 17. Amethod for growing cells, comprising: a) providing flexible tubingcontaining culture medium, and a surface in which the living cells cangrow on in said tubing, and a system of clamps, each of the clamps beingcapable of open and closed positions, the clamps being positioned so asto be able to divide the tubing into: i) an upstream region containingunused culture medium; ii) a downstream region containing spent culturemedium; and iii) a growth chamber region for growing said cells disposedbetween the upstream and downstream regions; and b) closing selectedones of the clamps on the tubing to define the growth chamber region ofthe tubing between the upstream and downstream regions of the tubing,and introducing viable cells into the growth chamber region; c)cyclically closing and opening selected ones of the clamps to redefinethe growth chamber region of the tubing so that a first portion of thepreviously defined growth chamber region becomes a portion of thedownstream region of the tubing, and a portion of the previously definedupstream region of the tubing becomes a portion of the growth chamberregion of the tubing; and d) repeating step c) until a sufficient amountof cells have been grown.
 18. The method according to claim 17,comprising the further step of withdrawing a sample of living cells fromsaid culture medium from the downstream region.
 19. The method accordingto claim 17, further comprising isolating said living cells from thedownstream region.
 20. The method according to claim 17, wherein theliving cells are selected from the group consisting of yeast cells,animal cells, plant cells, and stem cells.
 21. The method according toclaim 17, wherein the living cells are stem cells.
 22. The methodaccording to claim 21, wherein the living cells are selected from thegroup consisting of hematopoietic stem cells, bone marrow stem cells,stromal cells, astrocytes, oligidendrocytes, embryonic stem cells, fetalstem cells, umbilical cord stem cells, placenta derived stem cells, andadult stem cells.
 23. The method according to claim 22, wherein theliving cells are selected from the group consisting of hematopoieticstem cells, bone marrow stem cells, stromal cells, astrocytes andoligidendrocytes.
 24. The method according to claim 17, wherein thesurface in which the cells are grown is the interior surface of thetubing.
 25. The method according to claim 17, wherein the surface inwhich the cells are grown is a continuous fiber.
 26. The methodaccording to claim 17, further comprising growing the cells in one ormore growth chambers that are present in the growth chamber region. 27.The method according to claim 17, further comprising growing one or moretypes of cells in the growth chamber region.
 28. The method according toclaim 17, wherein the sufficient amount of cells of step d) is definedas a pre-determined density level of the cells.
 29. The method accordingto claim 17, wherein the tubing is gas permeable
 30. The methodaccording to claim 17, wherein the tubing is gas impermeable.
 31. Themethod according to claim 17, wherein the tubing is one of transparentand translucent, a turbidimeter being used to determine the densitylevel of the cells.
 32. The method according to claim 17, furthercomprising regulating the pressure of the growth chamber portion of thetubing relative to ambient pressure.
 33. The method according to claim17, further comprising measuring a pH of the culture medium in thegrowth chamber region.
 34. The method according to claim 17, furthercomprising regulating the temperature of the growth chamber region witha temperature regulator constructed to control the temperature of thegrowth chamber region of the tubing.
 35. The method according to claim17, further comprising agitating the culture medium in the growthchamber region with an agitator.
 36. The method according to claim 35,wherein the agitator comprises at least one stirring bar.
 37. The methodaccording to claim 17, further comprising subjecting the growth culturechamber region to at least one of radio waves, light waves, x-rays,sound waves, an electro magnetic field, and radioactive field.
 38. Themethod according to claim 17, further comprising subjecting the growthchamber region to a different gravitational force.